Project Summary Zika virus (ZIKV) is a mosquito-borne flavivirus and has historically been reported to cause mild symptomatic diseases and neurological syndromes during human infections. More recently, the explosion of microcephaly among infants born to ZIKV-infected women has made ZIKV a global public health concern. Apart from reports of sexual and congenital transmission, ZIKV is primarily transmitted by certain infected species of Aedes mosquitoes. In the absence of a vaccine or an approved antiviral drug, vector control strategies remain the only mechanism for control of Zika infections. While ZIKV causes acute human diseases, infections of vector mosquitoes are basically non-pathogenic, allowing persistent infections and conferring lifelong ability to transmit the virus. However, the mechanisms of establishment and maintenance of persistent infections in mosquitoes are unclear. In addition, vertical transmission of flaviviruses, which plays an unknown role in maintaining the virus in nature during adverse conditions, is poorly understood. The objective of this application is to elucidate the mechanisms of persistent infections in mosquitoes, with the ultimate goal of developing new strategies for transmission interventions. Research over the past several years on various mosquito-borne viruses has indicated that the different outcomes of arboviral infections in humans and mosquitoes are due to differences in the innate-immune responses between the two hosts. ZIKV, like most arboviruses, contains RNA as its genetic material, and several studies have suggested that the RNA- interference (RNAi) defense pathway is a major antiviral defense mechanism in mosquitoes. Nevertheless, it is unclear how ZIKV evades the robust RNAi response to establish persistent infections. Recent published studies have indicated that DNA forms of several arboviral RNA genomes are generated following infection in mosquito cell cultures and in mosquitoes, and small RNAs (sRNAs) deriving from viral DNAs (vDNAs) reinforce the RNAi response against viral infections. The proposed work tests a hypothesis that upon infection, vDNA in somatic cells aids to establish and maintain persistent infections, whereas vDNA in germline cells restricts virus replication. The Specific Aims are to determine I) the role of vDNA in ZIKV replication using mosquito cell cultures, and II) the role of vDNA in ZIKV replication in germline cells. Mosquito cell lines Aag and U4.4 from Aedes aegypti and Aedes albopictus, respectively, will be used for Aim I and colonized populations of both species will be used for Aim II. The results of these studies are expected to shed light on mosquito-virus interactions and lead to the development of new vector-control strategies. For example, it may be possible to develop strategies to stop the transition from acute to persistent infections in mosquitoes and/or to eliminate the infected mosquitoes from the population.